1. Field of the Invention
This invention relates to biochemical sensors for use in detecting analytes. In particular, the invention relates to self-referencing enzyme-based microsensors for measuring the flux of electrochemically inactive analytes.
2. Description of the Related Art
Glucose transport is a common and important component of cell metabolism. Studying glucose transport in pancreatic xcex2-cells is particularly interesting, because these cells play a role in insulin secretion and Type II diabetes. In pancreatic xcex2-cells, the movement of glucose from the blood across the plasma membrane is a key component in controlling insulin secretion, the chemical regulator of blood glucose concentration. Thus, following the glucose transport process in pancreatic xcex2-cells can provide important insights into the mechanisms underlying insulin secretion and Type II diabetes. Central to the control of insulin secretion is the concept of a cellular glucose sensor and its regulatory role. Further, down-regulation of the glucose transporter in pancreatic xcex2-cells correlates with decreased glucose uptake and the loss of insulin secretion in Type II diabetes. In order to gain further understanding of glucose transport, insulin secretion, and Type II diabetes, it would be desirable to measure changes in the glucose microenvironment surrounding pancreatic xcex2-cells non-invasively and in real time.
Despite a strong interest in tracking glucose transport and consumption, there has been no method available for the real-time measurement of glucose flux. Glucose consumption has been measured using, for example, the radiochemistry of 3H2O production from D-[5-3H] glucose metabolism (Guillam et al., Diabetes 49:1485-1491 (2000)), 14CO2 production from [14C] glucose oxidation (McDaniel et al., Diabetologia 10:303-308 (1974)), or microfluorometry of glucose and 2-deoxyglucose uptake using a cocktail of hexokinase, ATP, glucose-6-phosphate and NADP (Wree, Eur. J. Morhphol. 28:132-138 (1990)). Such techniques employing radiochemistry are hazardous and time consuming, and cannot be used with living tissues to provide real-time flux measurements.
Glucose and many other biologically important molecules such as, for example, lactate and glutamate, are not electrochemically active. Such molecules do not readily undergo reaction at an electrode to produce a detectable current, and therefore they cannot directly be measured electrochemically. However, many such molecules are acted upon by enzymes to generate electrochemically active species that are detectable by an electrode. For example, the processing of glucose by the enzyme glucose oxidase (GOx) generates hydrogen peroxide, which is easily detectable electrochemically. The process, which requires oxygen as a co-substrate, is outlined in Equations (1) and (2). Similar transformations are performed on lactate and glutamate by lactate oxidase and glutamate oxidase, respectively.                     glucose        +                              O            2                    ⁢                      ⟶            GOx                    ⁢          gluconolactone                +                              H            2                    ⁢                      O            2                                              (        1        )            
The production of H2O2 is measured at a charged platinum electrode surface:                                           H            2                    ⁢                                    O              2                        ⁢                          ⟶                                                +                  600                                ⁢                mV                                      ⁢                          O              2                                      +                  2          ⁢                      H            +                          +                  2          ⁢                      e            -                                              (        2        )            
Enzyme-based electrodes for indirectly detecting biologically important yet electrochemically inactive molecules such as glucose are well known in the art (e.g., U.S. Pat. No. 3,539,455). Glucose oxidase-based electrodes have been used to detect in vivo levels of circulating glucose (Hu et al., J. Neurochem. 68:1745-1752 (1997); Zhang et al., Anal. Chim. Acta, 281:513-520 (1993)), although in such applications the electrode is usually greater than 200 xcexcm in tip diameter. Miniaturized glucose microsensors are disclosed by Jung et al., J. Biol. Chem. 275:6642-6650 (2000). However, a need exists in the art for an enzyme-based electrode technique for non-invasively and accurately measuring the real-time flux of glucose and other electrochemically inactive analytes in a biological microenvironment.
Position modulation of microprobes has been used to enhance detection in various applications such as scanning electrochemical microscopy (Wipf et al., Anal. Chem. 64:1362-1367 (1992)), atomic force microscopy (Siedlecki et al., Biomaterials 19:441-454 (1998)), and scanning reference electrode techniques used in both biological and materials sciences (Nuccitelli, xe2x80x9cVibrating Probe Technique for Studies of Ion Transport,xe2x80x9d in Noninvasive Techniques in Cell Biology, pp. 273-310, Foskett et al. eds., Wiley-Liss, Inc., 1990; Kinlen et al., J. Electrochem. Soc. 146:3690-3695 (1999)). Position modulation enhances detection by self-referencing electrodes, which measure difference values between two positions, thereby reducing the impact of noise and drift, which are common to both positions. Self-referencing sensors have been used to measure the flux of ions (U.S. Pat. No. 6,062,225; Smith et al., Microsc. Res. Tech. 46:398-417 (1999); Smith et al., Am. J. Physiol. 280:C1-C11(2001)), as well as gases such as oxygen and nitric oxide (U.S. Pat. No. 5,968,340; Kumar et al., J. Neurosci. 21:215-220 (2001); Land et al., J. Exp. Biol. 202: 211-218 (1999)) in biological microenvironments.
The present invention depends, in part, upon the development of an enzyme-based microsensor capable of operating in self-referencing mode on a micron-scale to measure the real-time flux of an electrochemically inactive analyte in a biological microenvironment non-invasively and with reduced impact of sensor drift or noise. Fluxes on the order of pmol cmxe2x88x922 sxe2x88x921 can be measured using the methods and systems of the invention. For example, glucose fluxes as low as about 8 pmol cmxe2x88x922 sxe2x88x921 and glutamate fluxes as low as about 5 pmol cmxe2x88x922 sxe2x88x921 have been measured using self-referencing enzyme-based microsensors of the invention.
In one aspect, the invention provides a system for determining a flux of an analyte with respect to a source. The system comprises an electrode that includes an enzyme specific for the analyte. In the presence of the analyte, the enzyme generates a species detectable by the electrode. The system also comprises a translational mechanism for moving the electrode between at least two positions. At each of the positions, the concentration of the detectable species is dependent on the concentration of the analyte. The system can also comprise a voltage source for applying a voltage to a portion of the electrode at each of the two positions. Upon application of the voltage, the detectable species undergoes a chemical change to generate a position-dependent current. The system can also comprise sensing circuitry for detecting a current from the electrode in response to the applied voltage at each position. The system can also comprise analysis circuitry for converting the detected currents into a determination of flux of the analyte. The analysis circuitry can comprise a computer. The system can also comprise a controller for providing to the translational mechanism a frequency for the motion of the electrode. The controller can comprise a computer. The system can also comprise an oxygen source for preventing distortion of the flux determination by oxygen depletion.
In some embodiments, the source comprises a mammalian cell. In some embodiments, the enzyme is a dehydrogenase. In other embodiments, the enzyme is an oxidase. For example, the analyte and enzyme can be glucose and glucose oxidase, glutamate and glutamate oxidase, or lactate and lactate oxidase, respectively. In some embodiments, the detectable species is hydrogen peroxide.
In another aspect, the invention provides a system for determining a flux of each of a plurality of analytes with respect to a source. For each analyte, the system comprises an electrode including an enzyme specific for that analyte. In the presence of each analyte, the corresponding enzyme generates a species detectable by the electrode. The system also comprises a translational mechanism for moving each of the electrodes between at least two positions. At each of the positions, the concentration of the detectable species is dependent on the concentration of the analyte. The system can also comprise a voltage source for applying a voltage to a portion of each of the electrodes at each of the two positions. Upon application of the voltage, each of the detectable species undergoes a chemical change to generate a position-dependent current. The system can also comprise sensing circuitry for detecting a current from each of the electrodes in response to the applied voltage at each position. The system can also comprise analysis circuitry for converting the detected currents to a determination of flux for each of the analytes.
In another aspect, the invention provides a method of determining a flux of an analyte with respect to a source. The method comprises oscillating an electrode including an enzyme between first and second positions proximal to the source. The enzyme contacts the analyte and generates a species detectable by the electrode. At each of the positions, the concentration of the detectable species is dependent on the concentration of the analyte. A voltage is applied to a portion of the electrode so that the detectable species undergoes a chemical change to generate a position-dependent current. The current is sensed at the first and second positions and the sensed currents are used to determine the flux of the analyte with respect to the source. The method can also comprise providing oxygen to the source to prevent distortion of the flux determination by oxygen depletion.
In some embodiments, the source comprises a mammalian cell. In some embodiments, the enzyme is a dehydrogenase. In other embodiments, the enzyme is an oxidase. For example, the enzyme and analyte can be glucose oxidase and glucose, glutamate oxidase and glutamate, or lactate oxidase and lactate, respectively. In some embodiments, the detectable species is hydrogen peroxide. In such embodiments, the method can also comprise contacting the source with catalase to prevent distortion of the flux determination near the source.
In some embodiments, the oscillating step includes moving the electrode at a frequency in the range of about 0.1 Hz to about 0.3 Hz. In some embodiments, the distance between the first and second positions is between about 10 xcexcm and about 50 xcexcm. In some embodiments, the first and second positions lie within a gradient of the analyte with respect to the source. In some embodiments, the distance between the surface of the source and the midpoint between the first and second positions is between about 2 xcexcm and about 500 xcexcm.
In another aspect, the invention provides a method of assessing the viability of an embryo by determining a flux of an analyte with respect to the embryo. The method comprises oscillating an electrode including an enzyme between first and second positions proximal to the embryo. The enzyme contacts the analyte and generates aspecies detectable by the electrode. At each of the positions, the concentration of the detectable species is dependent on the concentration of the analyte. A voltage is applied to a portion of the electrode so that the detectable species undergoes a chemical change to generate a position-dependent current. The current is sensed at the first and second positions. The sensed currents are used to determine the flux of the analyte with respect to the embryo, and the flux determination is used to assess the viability of the embryo.
In still another aspect, the invention provides a method of screening for compounds that affect a transmembrane flux of an analyte with respect to a cell. The method comprises oscillating an electrode including an enzyme between first and second positions proximal to the cell. The enzyme contacts the analyte and generates a species detectable by the electrode. At each of the positions, the concentration of the detectable species is dependent on the concentration of the analyte. A voltage is applied to a portion of the electrode so that the detectable species undergoes a chemical change to generate a position-dependent current. The current is sensed at the first and second positions and the sensed currents are used to determine the transmembrane flux of the analyte with respect to the cell. The cell is then contacted with a test compound. The oscillating, voltage application, current sensing, and flux determination steps are repeated for the cell in the presence of the test compound. The transmembrane flux of the analyte with respect to the cell in the presence of the test compound is compared to the transmembrane flux of the analyte with respect to the cell in the absence of the test compound. A difference in flux values indicates that the test compound affects the transmembrane flux of the analyte with respect to the cell.